Project Summary/Abstract Chronic infection with hepatitis B virus (HBV) affects approximately 350 million people worldwide and constitutes a significant risk factor for severe liver disease, including hepatocellular carcinoma (HCC). The HBV regulatory HBx protein is required for virus replication, but a specific role for HBx in the replication pathway remains to be identified. Several HBx-interacting proteins have been identified, but their role in HBV replication has not been investigated. The interaction of HBx with cellular DDB1 was identified in our laboratory using the yeast two-hybrid assay, and this interaction is important for virus replication. Although HBx has been extensively studied as a broadly acting transcriptional transactivator, in this proposal we hypothesize that an important role for HBx in virus replication may occur via post-translational regulation of cellular proteins. The objective of this research is to define the role of HBx in virus replication and pathogenesis, and this will be studied in three specific aims. In Specific Aim 1, we will screen a panel of HBx mutant proteins for their ability to restore HBx-deficient HBV replication. Conversely, the ability of replication- defective mutants to inhibit wildtype replication will be studied. A role for other HBx binding partners in HBV replication will be determined in gene silencing experiments. In Specific Aim 2, the effect of DCAFs (DDB1-binding proteins) on virus replication will be investigated. A role of the 26S proteasome in virus replication will be examined. Experiments will address whether HBx can modify the spectrum of DDB1-binding partners in Lentivirus-X-transduced cells. In Specific Aim 3, the hydrodynamic model of HBV replication will be studied in DDB1 knockdown mice. The effect of HBx on the acute phase response will be investigated using the hydrodynamic model. These experiments will identify virus-cell interactions that are essential for HBV replication. Our studies will provide insight into the underlying mechanism by which HBx promotes virus replication, with the opportunity of identifying novel targets through which to disrupt ongoing viral replication.